The goal of this project is to characterize and control novel epigenetic regulatory mechanisms that drive altered gene activity in cancer. The majority of our initial efforts in this area have focused on the study of protein and nucleic acid acetylation. Using a suite of novel chemical tools that allow us to study acetylation mechanisms directly in cells, we have discovered new enzymatic and non-enzymatic acetylation pathways that are highly elevated in cancer. In addition, we have made substantial progress characterizing the activity, druggability, and metabolic regulation of these mechanisms. These advances are grouped according to four specific aims. 1. Discovery and characterization of novel acetyltransferase enzymes. Chemoproteomic profiling methods pioneered in our lab led to the discovery of a relatively uncharacterized acetyltransferase, NAT10, which is highly upregulated in a variety of cancer cell lines, and also sensitive to the metabolic state of the cell. Subsequent work has revealed the primary function of NAT10 is the catalysis of RNA cytidine acetylation, which evidence suggests extends to diverse elements of the transcriptome including ribosomal RNA, transfer RNA, and messenger RNA . Over the past year we have developed multiscale chemical approaches to biologically, biophysically, and biochemically characterize cytidine acetyltransferase activity, including the development of a method for directly sequencing substrates of these enzymes, which we continue to apply towards the goal of defining the role of NAT10 and its homologues in acetylation-dependent signaling. Beyond NAT10, we continue to extend our chemoproteomic profiling technology and integrate it with cutting-edge systems biology analysis tools to enable the discovery of new enzymatic and non-enzymatic acetylation mechanisms involved in cancer. 2. Characterization of acetyltransferase inhibitors. Targeting the cellular acetylation machinery is an emerging paradigm in oncology. However, relatively few small molecule inhibitors of acetyltransferases are known. To address this unmet need, our group has developed biochemical, chemoproteomic, and cell-based assays that can be used to unambiguously interrogate the activity of small molecule acetyltransferase inhibitors. These methods enabled the first evidence for cellular occupancy and on-target activity of a small molecule lysine acetyltransferase inhibitor. Currently we are applying these approaches in collaboration with industry to define the selectivity of novel classes of acetyltransferase inhibitors. In addition, we continue to apply these methods to characterize the pan-assay interference features of reported acetyltransferase inhibitors, which has proved critical in moving the field forward by aiding the interpretation of the activity of these molecules in cellular assays. 3. Metabolic regulation of epigenetics. Emerging evidence indicates that metabolism itself may function as an epigenetic mechanism, through the ability of metabolites to modulate the activity of enzymes involved in epigenetic and epitranscriptomic regulation of gene expression, as well as directly react with amino acid residues leading to the deposition of non-enzymatic protein posttranslational modifications. Our previous work has focused on the development of technologies for studying the metabolic regulation of acetylation. In the past year, In the past year, we have extended these methods to study the mechanism of action of oncometabolites, a class of cancer metabolites that can directly trigger tumorigenic signaling. Using the oncometabolite fumarate which accumulates in the hereditary cancer syndrome predisposition HLRCC as an initial model, we have shown that chemoproteomic methods can be used to identify hotspots of oncometabolite reactivity and quantify their level of protein modification on a proteome-wide scale. This approach led to the identification of a novel reactivity motif as well as new tumor suppressor targets of fumarate. Our current studies are focused on understanding the precise input of oncometabolite reactivity into gene regulation, extending these methods to other classes of covalent and noncovalent metabolites, and testing whether insights from chemoproteomics can be used to guide new therapeutic approaches. 4. Diagnostic detection of oncometabolites. Our group has pioneered the use of fluorogenic alkene-nitrileimine cycloadditions to detect fumarate, an oncometabolite that accumulates to high levels in HLRCC (see above). Very recently we reported a novel chemical scaffold (diaryl tetrazole) that greatly improve the sensitivity of this approach, and demonstrated it could be applied to detect aberrant fumarate hydratase (FH) activity in tumor xenografts, as well as to image this oncometabolite directly in living cells. In our current studies we are applying this in flow cytometry-based in combination with metabolism-focused CRISPR-Cas9 knockout screens to identify novel pathways that may drive (or rescue) elevated fumarate levels in cancer, as well as exploring whether oncometabolite reactivity may be leveraged for immunodetection of HLRCC. These combined aims leverage new cellular enzyme profiling technologies to discover novel epigenetic mechanisms, validate next-generation therapeutics, and develop novel diagnostics for cancer treatment.